Despite multiple changes in chemotherapy over the past 15-20 years, the 2-year metastasis-free survival for Ewing's sarcoma remains at 40% with a 3-year overall survival of 50%. Understanding the mechanisms that contribute to the growth of Ewing's sarcoma may assist in the development of new therapeutic approaches. We demonstrated that Ewing's tumor cell lines and primary patient tumor specimens overexpress VEGF with a shift from the membrane-bound 189 isoform to the soluble 165 isoform. VEGF165 has been shown to chemoattract stem cells. Using our Ewing's sarcoma nude mouse model in a transplant setting we demonstrated that bone marrow (BM) cells migrate into the tumor, differentiate into endothelial cells and contribute to the formation of the tumor vasculature. We demonstrated that migrated cells co-localize with tumor vessels between the tumor/normal border and that VEGF165 is responsible for the chemoattraction of these stem cells. This activity was not duplicated by VEGF189. Further investigations using VEGF165-siRNA support our hypothesis that these migrated stem cells play an important role in tumor growth and tumor vessel development. Taken together our data indicate that vasculogenesis, in addition to angiogenesis is involved in the expansion of the tumor vasculature contributing to about 10% of the vessels during the first week of tumor growth. We wish to continue investigating the vasculogenesis process by identifying the stem cell subpopulations that participate, determining the importance of this process in both primary and metastatic tumor growth and defining the mechanism(s) by which stem cells are attracted to specific tumor vascular areas. We propose to (1) define the BM cell subpopulations that contribute to the vasculogenesis process in Ewing's sarcoma; (2) determine whether BM stem cells participate in the growth and development of Ewing's metastases in either the bone or the lung; (3) determine the importance of vasculogenesis to tumor growth and whether vasculogenesis can restore tumor growth and vessel development in the absence of VEGFies', (4) determine whether NOTCH signaling, in specific DLL4, plays a role in the chemoattraction of stem cells to the Ewing's tumor. Understanding how vasculogenesis contributes to the growth and metastasis of Ewing's sarcoma and ascertaining the chemotactic signals involved in this process may open up new therapeutic approaches to treat patients with this disease.